CN1301267C - A simulated epitope peptide of MUC1 mucoprotein and its encoding DNA and use thereof - Google Patents
A simulated epitope peptide of MUC1 mucoprotein and its encoding DNA and use thereof Download PDFInfo
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- CN1301267C CN1301267C CNB2005100776174A CN200510077617A CN1301267C CN 1301267 C CN1301267 C CN 1301267C CN B2005100776174 A CNB2005100776174 A CN B2005100776174A CN 200510077617 A CN200510077617 A CN 200510077617A CN 1301267 C CN1301267 C CN 1301267C
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Abstract
The present invention discloses a simulated epitope peptide of MUC1 mucoprotein, and coding DNA and applications of the peptide. The present invention aims at providing a simulated epitope peptide of MUC1 mucoprotein, coding DNA thereof, and the applications of the simulated epitope peptide to the preparation of vaccines and medicine for resisting tumors. The simulated epitope peptide is a polypeptide with one of the following amino acid residue sequences: 1) SEQ ID No. 1 in a sequence table and 2) a polypeptide with specific cells for inducing the generation of MUC1 mucoprotein and the function of humoral immune response, wherein the 2) polypeptide is formed by replacing or deleting or adding one to ten amino acid residue of the amino acid residue sequence of the SEQ ID No. 1. The coding DNA is one of the following nucleotide sequences: 1) a DNA sequence of SEQ ID No. 2 in the sequence table and 2) a nucleotide sequence which can form hybridization with the DNA sequence limited by the SEQ ID No. 2 in the sequence table under high precise conditions. The peptide of the present invention can be used as active ingredients for preparing vaccines or medicine, and can perform the functions for restraining or clearly expressing tumour cells of MUC1. The present invention has wide application prospect in the field of medicine.
Description
Technical field
The present invention relates to bio-pharmaceuticals and genetically engineered field, particularly relate to mucinous analogue epi-peptide of MUC1 and coding DNA thereof and its application in preparation anti-tumor vaccine and medicine.
Background technology
Tumor vaccine is a kind of means of oncobiology treatment, the existing last 100 years history of its research.Boon in 1991 etc. have successfully separated people's malignant melanoma antigen MAGE-1 of CTL specific recognition first, for new milestone (Boon etc., A gene encoding an antigen recognized bycytolytic T lymphocytes on a human melanoma.Science.1991Dec13 have been founded in the research of tumor vaccine; 254 (5038): 1643-7.).Tumor vaccine mainly is to break body by induce immune response the immunological tolerance of tumour cell is worked.Mainly comprise as the immunogen of tumor vaccine: complete tumour cell, tumor associated antigen, synthetic carbohydrate antigen, tumor antigen peptide from body or heterology tumour cell, modification, tumour antigen stimulates or dendritic cell, antiidiotypic antibody and derivative thereof, recombinant virus or the bacterial vaccine and the nucleic acid vaccine etc. of transfection.There are a plurality of tumor associated antigens to be used to the preparation of tumor vaccine at present.
MUC1 Saliva Orthana (hereinafter to be referred as MUC1) is a kind of high molecular of being expressed in cell surface (＞200kD) membrane glycoprotein, have two essential characteristics: (1) sugar chain content accounts for more than 50% of full molecular weight, how is connected with glycosylation site on the polypeptide backbone with O type glycosidic link; (2) contain 20-125 in the polypeptide backbone by " HGVTSAPDTRPAPGSTAPPA " totally 20 continuous tumor-necrosis factor glycoproteins (variablenumbers of tandem repeats that amino-acid residue is formed, VNTRs), all O type glycosylation sites all are arranged in VNTRs.Studies show that MUC1 is unconventionality expression in multiple epithelial origin malignant tumour (mammary gland, pancreas, ovary, colon, lung, stomach, bladder, kidney, oesophagus), mainly shows: (1) expression amount increases: more normally can improve more than 100 times; (2) expressive site changes:, promptly be the top and express (apical expression) in nearly tube chamber of cell or lumen of gland one side at normal tissue expression, do not contact with body immune system, and in tumour, whole cell surface is all expressed, and therefore, can contact with immunity system; (3) textural anomaly: incomplete mainly due to glycosylation, new sugar chain and peptide epitopes appear.
From the mid-80, but (Finn etc. since the CTL cell of existence specific recognition tumour MUC1 in discovery such as the Finn tumour patient body, Specific, major histocompatibility complex-unrestrictedrecognition of tumor-associated mucins by human cytotoxic T cells.Proc NatlAcad Sci USA.1989Sep; 86 (18): 7159-63.), MUC1 is a focus molecule of tumor vaccine research always, tumor vaccine with the MUC1 preparation mainly contains polypeptide vaccine, dna vaccination, dendritic cell vaccine, recombined vaccinia virus vaccine, sugar chain vaccine etc., and it is clinical that the research of some vaccine has entered the I/II/III phase.Studies show that MUC1 sugar chain knurl seedling is mainly induced the generation humoral immunoresponse(HI) in vivo; Though the MUC1 polypeptide vaccine can induce stronger CTL to reply in animal model, mainly based on humoral immunoresponse(HI), and autoimmune phenomena appears in indivedual case in human body.In addition, contain the MUC1 albumen of higher level in the tumour patient circulation of blood, can play sealing process the antibody that injects intravital monoclonal antibody or knurl generation that seedling is induced, thus the curative effect of greatly reducing.
Therefore, prepare new tumor vaccine, will help to address the above problem based on the MUC1 molecule.Mimic epitopes (mimotopes) peptide vaccine is exactly wherein a kind of.Mimic epitopes is meant the diverse two kinds of materials of primary structure owing to have identical space conformation, can combine with the antigen binding domain of same antibody or T cell surface receptor.Mimic epitopes can be simulated the antigenic determinant of various natural antigens, and at present commonly used have two kinds of polypeptide simulated albumin epi-position and polypeptide simulation polysaccharide or glycolipid epi-positions.Compare with natural epi-position, mimic epitopes has following advantage in theory: 1. also can obtain mimic epitopes and be used for prevention and treatment under natural epi-position condition of unknown; 2. by molecular modification, can strengthen combining of mimic epitopes and MHC molecule, more help the lymphocytic activation of T; 3. mimic epitopes is generally non-oneself protein, is expected to strengthen the immunne response of human body to former epi-position, or breaks the immune tolerance state [131415] to former epi-position.Studies show that recently, can in transgenic mice and human body, induce specificity cellular immunity response with the mimic epitopes of tumour antigen as immunogen, and not find autoimmune phenomena.
The acquisition of mimic epitopes has several different methods, and wherein the phage random peptide library of setting up based on display technique of bacteriophage is one of technology of at present the most frequently used screening mimic epitopes.Its basic ideas are: the random oligonucleotide sequence of chemosynthesis and phage capsid protein III or VIII gene are merged, and the small peptide that each seed amino acid is arranged is expressed in different phage surfaces, makes up phage random peptide library; Eluriate (biopanning) by biology and filter out the small peptide part that can combine with antibody or lymphocytic cell surface acceptor, the encoding sequence that inserts is identified in order-checking, obtains the aminoacid sequence of mimic epitopes.
Summary of the invention
The purpose of this invention is to provide mucinous analogue epi-peptide of MUC1 and coding DNA thereof.
The mucinous analogue epi-peptide of MUC1 provided by the present invention, name is called M1, derives from M13 phage (PhageM13), is the polypeptide with one of following amino acid residue sequences:
1) the SEQ ID № in the sequence table: 1;
2) with SEQ ID № in the sequence table: 1 amino acid residue sequence is through replacement, disappearance or the interpolation of one to ten amino-acid residue and have the polypeptide of inducing MUC1 Saliva Orthana specific cell and humoral immunoresponse(HI) effect.
SEQ ID № in the sequence table: 1 is made up of 12 amino-acid residues.
The present invention provides code book invention MUC1 the DNA (M1) of mucinous analogue epi-peptide M1 simultaneously, and it is one of following nucleotide sequence:
1) SEQ ID № in the sequence table: 2 dna sequence dna;
2) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 2 dna sequence dnas hybridization that limit.
The rigorous condition of described height can be that (or 0.1 * SSC), the solution of 0.1%SDS is hybridized and washed film with 0.1 * SSPE under 65 ℃.
SEQ ID № in the sequence table: 2 by 36 based compositions, and coding has SEQ ID № in the sequence table: the protein of 1 amino acid residue sequence.
The expression vector, transgenic cell line and the host bacterium that contain DNA of the present invention all belong to content of the present invention.
Arbitrary segmental primer is to also being content of the present invention among the amplification M1.
Another object of the present invention provides a kind of method of expressing the mucinous analogue epi-peptide of above-mentioned MUC1.
The method of the mucinous analogue epi-peptide of the above-mentioned MUC1 of expression provided by the present invention is that the recombinant expression vector that will contain the mucinous analogue epi-peptide coding DNA of above-mentioned MUC1 imports host cell, expresses obtaining the mucinous analogue epi-peptide of MUC1.
But described host is the protokaryon or the eukaryotic cell of arbitrary expression alien gene.
Described prokaryotic cell prokaryocyte can be colibacillus, as E.coli BL21, E.coliM15, E.coli JM109 or E.coliDH5 α etc.
Described eukaryotic cell can be mammalian cell, yeast cell and vegetable cell etc.Comprise COS-7, CHO, BHK-21 or Pichia pastoris etc.
The carrier that sets out that is used to make up the recombinant expression vector that contains the mucinous analogue epi-peptide coding DNA of MUC1 can be pET series, pGEX series, pMAL series, pBluescript SK-, pTrx, pTrxFus, pQE-30, pCAT3 series, p β-gal series, pcDNA3.1 (+/-), pCMV series, pCMVScript, pd2EGFP series, pGL3 series, pIND (SP1)/V5-HisA, pTET-Splice, pVgRXR or pPIC series etc.
Cultivation contains the substratum and the culture condition of the host cell of the mucinous analogue epi-peptide coding DNA of MUC1 of the present invention, all can be substratum and the culture condition of cultivating the host that sets out.
The present invention also provides a kind of anti-tumor vaccine and a kind of antitumor drug.
Anti-tumor vaccine provided by the present invention and antitumor drug, activeconstituents are the mucinous analogue epi-peptide of above-mentioned MUC1.
When needing, in above-mentioned antitumor drug, can also add one or more pharmaceutically acceptable carriers.Described carrier comprises thinner, vehicle, weighting agent, tackiness agent, wetting agent, disintegrating agent, absorption enhancer, tensio-active agent, absorption carrier of pharmaceutical field routine etc.
Medicine of the present invention can carry out combined therapy with microbiotic, immunostimulant etc., and can strengthen specificity cellular immunity response by selecting suitable antigen presenting cell (as dendritic cell), adjuvant or cytokine (as IL-2) combined utilization.
Medicine of the present invention can be made various ways such as injection liquid, tablet, pulvis, granula, capsule, oral liquid.The medicine of above-mentioned various formulations all can be according to the ordinary method preparation of pharmaceutical field.
The consumption of said medicine is generally the mucinous analogue epi-peptide of 0.5-2mg MUC1/kg body weight/day, and be 10-20 days the course of treatment.
The analogue epi-peptide M1 that the present invention obtains by MUC1 monoclonal antibody screening phage random peptide library, can be used as that active ingredient is prepared into vaccine or medicine is used in the body, induce cellullar immunologic response or humoral immunoresponse(HI), suppress or remove the tumour cell of expression MUC1 at MUC1.The present invention also can unite use with other medicines, prevention and treatment kinds of tumors disease.The present invention has broad application prospects at field of medicaments.
Description of drawings
Fig. 1 is 100,200,400,800 μ g/mL M1 peptides mix back and breast cancer cell MCF7 respectively with the Ma695 monoclonal antibody a competitive inhibition detected result for concentration.
Fig. 2 combines result of experiment for flow cytometer detects immune serum with the MUC1+ tumour cell.
Fig. 3 is a M1 peptide specific T cell proportion experimental result in the ELISPOT technology for detection immune mouse spleen lymphocyte.
Fig. 4 for be target cell with BST739, BST739-MUC1, to imitate the target ratio be 50: 1,100: 1 o'clock the molten broken rate statistics of target cell.
Fig. 5 respectively organizes tumour size comparative result for the tumor prevention model.
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment.All examining orders are finished by Shanghai biotechnology Services Co., Ltd.
The acquisition of the mucinous analogue epi-peptide M1 of embodiment 1, MUC1
Phage random 12 peptide storehouses (Ph.D.-12): available from New England Biolabs company.
One, the biology in Ph.D.-12 peptide storehouse is eluriated (Biopanning)
Selection can be discerned the Ma695 monoclonal antibody (available from China fir Bioisystech Co., Ltd in Beijing) of tumour MUC1 glycopeptide epi-position and as target molecule phage random 12 peptide storehouses be screened, and obtains the analogue epi-peptide M1 of MUC1, and concrete grammar is as follows:
First round screening: with coating buffer (0.1M NaHCO
3, pH8.6) concentration with the Ma695 monoclonal antibody is adjusted to about 10 μ g/mL, and every hole adds the above-mentioned antibody liquid coated elisa plate of 100 μ L, places 12-24 hour in 4 ℃, wet box; Discard coating buffer, firmly clap debris, confining liquid (0.1M NaHCO is filled it up with in every hole
3(pH8.6), 1%BSA), to place 1 hour for 4 ℃, discard confining liquid, wash fast 6 times with TBST (TBS+0.1%Tween-20 (V/V)). debris is fallen in each bat, does not make orifice drying; Dilute 10 μ L (4 * 10 with 100 μ L TBST
10Pfu) phage library liquid adds in the hand-hole, room temperature jog 60min; Discard unconjugated phagocytosis body fluid, clap debris; Wash 10 times with TBST, each 6min, and with suction pipe acutely piping and druming up and down; Add 100 μ L elutriants (0.2M Glycine-HCl, pH2.2,1mg/ml BSA), incubated at room 8min moves on to elutriant in the 1.5mL centrifuge tube, add immediately 30 μ L neutralizers (1M Tris-HCl, pH9.1), mixing; Learn from else's experience 10
-1, 10
-2, 10
-3, 10
-4Each 10 μ L of the elutriant of gradient dilution survey titre.The residue elutriant increases, and method is the same.
Second-four-wheel screening: the same first round of method, add 2 * 10
11The pfu phage of increasing, the concentration of Tween-20 is brought up to 0.5% (V/V) among the TBST.
Two, the amplification of plaque
The intestinal bacteria XL1-blue that learns from else's experience and cultivated 12-24 hour, dilute (containing 10 μ g/mL Tet (Sigma)) in 1: 100 ratio with LB, add the diluted bacterium liquid of 1mL in each test tube, get locus coeruleus after titre is measured in single four-wheel amplification back with the rifle choicest, be inoculated in the above-mentioned LB substratum that contains 10 μ g/mL Tet, 37 ℃ of 260rpm cultivate 4.5h; After cultivating end, with 4 ℃ of centrifugal 30sec of 10000rpm of nutrient solution, get the supernatant repeated centrifugation once, get 80% supernatant, 4 ℃ are preserved or add glycerine to final concentration is that 50% back is in-20 ℃ of preservations.
Three, with the ELISA method phage clone that obtains is analyzed
Ma695 monoclonal anti body and function coating buffer is diluted to 10 μ g/mL, the two removable enzyme plates of bar of bag quilt, every hole 100 μ L, parallel work two holes, 4 ℃ left standstill 12-24 hour; Discard coating buffer, confining liquid is filled it up with in every hole, 37 ℃ of sealing 1h; Wash 6 times with TBST (containing 0.5%Tween-20, down together), get the phage clone of step 2 after amplification, each gets 50 μ L+150 μ L TBST, and mixing adds in the plate hole, gets the former storehouse of 2 μ L+200 μ L TBST simultaneously in contrast, RT jog 1h; Wash 8 times with TBST, every hole adds with HRP/AntiM13 monoclonal antibody (amercham pharmacia biotech) the 100 μ Ls of confining liquid (containing 1mg/mL BSA) by 1: 5000 dilution proportion, and room temperature jog 1h, TBST wash 8 times; Every hole adds 100 μ L TMB colour developing liquid, and 37 ℃ of 10-15min that develop the color down add 50 μ L 2MH again
2SO
4Color development stopping is measured OD450 value, and the result is as shown in table 1, selects and contrasts ratio and check order greater than 2 phage clone extraction ssDNA and analyze.
The elisa assay result of table 1 phage clone
| Numbering | O.D.450 | |
| The MUC1 phage | Negative control albumen | |
| 123456789 10 11 12 13 14 15 former storehouse contrasts | 0.087 0.096 0.092 0.111 0.088 0.083 0.082 0.101 0.088 0.097 0.122 0.139 0.117 0.098 0.123 0.066 | 0.043 0.043 0.041 0.042 0.042 0.041 0.038 0.038 0.041 0.031 0.052 0.062 0.054 0.053 0.043 0.065 |
Four, the acquisition of M1
Choose in conjunction with the strongest active phage clone and check order, derive peptide epitopes sequence with the Ma695 antibodies according to sequencing result, obtain 5 kinds altogether and respectively contain 12 amino acid whose peptide epitopes, with one of them called after M1, M1 has SEQ ID № in the sequence table: 1 amino acid residue sequence, SEQ ID № in the sequence table: 1 is made up of 12 amino-acid residues, and the DNA of coding M1 has SEQ ID № in the sequence table: 2 Nucleotide, the SEQID № in the sequence table: 2 by 36 based compositions.Select M1 to do further research with following method.
The competitive binding experiment of embodiment 2, M1 peptide, Ma695 and MCF7
With concentration is that 100,200,400,800 μ g/mL M1 peptides mix with the Ma695 monoclonal antibody that is 0.25 μ g/mL respectively through weaker concn, place 5min on ice, mix with breast cancer cell MCF7 (ATCC) again, place 45min on ice, again with the 2%PBA washed twice, the second antibody of fluorescein isothiocyanate (FITC) mark that adds dilution in 1: 100, is placed 45min on ice at lucifuge; After the PBS washed twice, PBS 200 μ l re-suspended cells with precooling, detect with flow cytometer, the fluorescence intensity detected result is (the positive cell count of ordinate zou as shown in Figure 1, X-coordinate is a relative intensity of fluorescence), show combining of M1 peptide competitive inhibition Ma695 and MCF7 cell, retarding effect has dose-dependently, when cell count is 2 * 10
5Individual, Ma695 concentration is 0.25 μ g/mL, when the M1 peptide concentration is respectively 100,200,400,800 μ g/mL, positive cell percentage is respectively 92.7%, 87.7%, 86.7% and 82.5%, and relative intensity of fluorescence is respectively 3.85,3.20,3.20,2.80.According to the relative intensity of fluorescence changing conditions, by formula calculate inhibiting rate: inhibiting rate=(fluorescence intensity before suppressing-inhibition back fluorescence intensity)/fluorescence intensity * 100% before suppressing.Inhibiting rate is respectively 11%, 26%, 26% and 34%, shows combining of M1 Toplink competitive inhibition Ma695 antibody and former epi-position, is real mimic epitopes.
1, uses dendritic cell (DC) immune mouse of load M1 peptide
The cultivation of spleen dendritic cell: the disconnected neck of T739 mouse is put to death, and the aseptic spleen of getting grinds, and crosses 200 order stainless steel filtering nets, makes single cell suspension; Splitting erythrocyte is after PBS washs 3 times, by 2 * 10
6The cell density of/ml adds plastic culture dish, abandons suspension cell after 4 hours, and attached cell adds DC special culture media and cytokine (IL-41000U/mL, GM-CSF 1000U/mL), the next day half amount change liquid; Collection in the 6th day suspends and loose attached cell changes in the new culture dish, and adding LPS (1 μ g/mL) stimulates the DC maturation.
Collect ripe DC, by concentration adding M1 peptide or the TAT-M1 of 10 μ g/mL, 37 ℃, 5%CO
2Cultivated 1 hour under concentration and the saturated humidity, be interrupted therebetween and shake for several times.After the washed twice, according to ripe DC purity, being adjusted to DC density with PBS is 2 * 10
6Individual/mL.4～8 ages in week, female T739 was divided into four groups at random, and every group 2-4, the ripe DC suspension (2 * 10 of tail vein injection 0.1mL
5/ mouse), or empty DC, or the PBS of respective volume.Weekly immunity once, totally three times; The immunity of TAT-M1 peptide once.
2, DC+PBS immune mouse
Method is replaced the M1 peptide with 1 with PBS.
3, PBS immune mouse
Method does not add DC with 2.
4, flow cytometer detects the situation that combines of immune serum and MUC1+ tumour cell (the phalangeal cell surface expression has the tumour cell of MUC1)
Get the eyeball of step 1-3 immune mouse and collect serum, three kinds of serum respectively by after 1: 20 dilution proportion with mouse source transitional cell bladder carcinoma cell line BST739 (the MUC1-mouse source transitional cell bladder carcinoma cell line BST739 (Jiang Feng that does not express MUC1, Zhou Xingming, Zhang Zhonglin etc. the foundation [J] of portable mouse transitional cell carcinoma of bladder model. Chinese magazine of urology surgery, 1993,14 (2): 184-186)), breast cancer cell MCF7 (MUC1+ breast cancer cell MCF7, ATCC) and prostate cancer cell PC-3 (MUC1+ prostate cancer cell PC-3, ATCC) and BST739-MUC1 (go into people MUC1 full-length cDNA (GenBank:J05582) at the BST739 transit cell, this cytotostatic is expressed MUC1) combination, contrast is normal mouse serum, detects positive cell number and relative intensity of fluorescence (fluorescein isothiocyanate (FITC) mark) with flow cytometer.(the positive cell count of ordinate zou, X-coordinate are relative intensity of fluorescence to the result as shown in Figure 2.1 is the mice serum after the DC immunity of load M1 peptide; 2 is the mice serum after the DC+PBS immunity.A is MUC1-mouse source transitional cell bladder carcinoma cell line BST739, B is MUC1+ breast cancer cell MCF7, C is MUC1+ prostate cancer cell PC-3, D is BST739-MUC1, behind the ripe DC immunity T739 mouse of load M1 peptide, mice serum can combine with MUC1+ breast cancer cell MCF7, MUC1+PC-3 and BST739-MUC1, but does not combine with MUC1-BST739; Combine (positive rate is respectively 26% and 13.5%) with DC+PBS immunity mice serum that obtains and MUC1+MCF7 that derives from the people and MUC1+PC-3 part, but do not combine with the MUC1-mouse source transitional cell bladder carcinoma cell line BST739 and the BST739-MUC1 in homology mouse source; Do not combine with the mice serum after the PBS immunity with above-mentioned any cell.
M1 peptide specific T cell proportion in embodiment 4, the ELISPOT technology for detection immune mouse spleen lymphocyte
Obtain the ripe DC of load M1 peptide with the method identical, and, be contrast (identical) with embodiment 3 with the DC+PBS mice immunized with its immune T739 mouse with embodiment 3; With with quadrat method with the immune T739 mouse of TAT-M1 peptide (chemosynthesis, HPLC purifying).With M1 peptide specific T cell proportion in the ELISPOT technology for detection immune mouse spleen lymphocyte (used ELISPOT detection kit available from Dutch U-Cytech company): dilute good coated antibody bag by 96 orifice plates with 50 μ L, add PBS to 100 μ L, seal with sticking paper and to avoid evaporating, hatched 12-24 hour for 4 ℃, abandon supernatant, PBST (PBS+0.05%Tween-20) washes 5-10 time, and each 1 minute, each washing back patted dry on thieving paper repeatedly.200 μ L confining liquids (PBS that contains 1%BSA) seal every hole, hatch 1h or 4 ℃ for 37 ℃ and hatch 12-24 hour.Abandon supernatant, do not wash.Every hole adds the monolayer cell suspension (antigenic stimulation 5 * 10 of 100 μ L preparation
4Individual cells/well).Cover lid, 37 ℃, 5%CO
2Hatch 5h under concentration and the saturated humidity, during keep the incubator vibrations, prevent to influence spot and form.Abandon supernatant, every hole adds the ice-cold deionized water of 200 μ L, places 10min on ice, and PBST washes 10 times, pats dry.Abandon supernatant, every hole adds the diluted vitamin H of 100 μ L and detects antibody, hatches 1h or 4 ℃ for 37 ℃ and hatches 12-24 hour.Abandon supernatant, PBST washes 5-10 time, pats dry.Every hole adds the diluted anti-biotin antibodies of 50 μ L (GABA), hatches 1h for 37 ℃.Abandon supernatant, PBST washes 5-10 time, pats dry (not wanting residual washing lotion in the hole).Every hole adds the freshly prepared developers of 30 μ L (adding) on ice, and place room temperature, dark place, hatches 15-30min.Microscopically is observed spot and is formed situation, when clear spot is visible, and tap water flushing termination reaction.Drying at room temperature 96 orifice plates, the every hole of ELISPOT stigmatic image analyser (BIOSYS product) counting spot number.The result judges: higher, the disperse gradually towards periphery of positive spots diameter central authorities' density; In each experiment, compare with negative control group, the spot number increases thinks meaningful more than 2 times.(A among the figure, A1, A2, A3 are parallel 4 holes of the same sample of M1 peptide experimental group to detected result as shown in Figure 3; B, B1, B2, B3 are parallel 4 holes of the same sample of empty DC control group), measure the quantity that spleen cell is secreted the M1 specific, activated T lymphocytes of gamma-interferon, measurement result (n is the mouse quantity of this group) as shown in table 1, external without antigenic stimulation, per 10
5The spot number of individual splenocyte is 52 ± 8.5 (also can be expressed as 52 ± 8.5/10
5Splenocyte); External after 10 μ g/mL M1 peptides stimulate 40h, the spot number obviously increases, and reaches 112.5 ± 16.2/10
5Splenocyte shows in the spleen cell, and the lymphocytic ratio of M1 peptide specific activated T is 0.11%; The external warp of mouse spleen lymphocyte or stimulate without the M1 peptide after the DC+PBS immunity, spot all is less than 10/10
5Splenocyte, similar with the PBC immune.
Table 2 ELISPOT detects the M1 specific, activated T lymphocytes of spleen cell secretion gamma-interferon
| 1，n＝4 | 2，n＝4 | 3，n＝6 | |||||
| DC+M1 | DC+PBS | DC+M1 | DC+PBS | DC+M1 | DC+TAT-M1 | DC+PBS | |
| Do not stimulate pre-stimulation | 52.5±8.5 112.5±16.2 | 4.5±2.1 6.5±2.1 | 61.5±9.2 105.5±13.4 | ＜10 ＜10 | 46.5±7.9 116.3±20.4 | 34.8±6.2 40.8±10.5 | ＜10 ＜10 |
The result is with per 10
5The positive spots numerical table that individual splenocyte forms shows.Classify result of experiment in the table as 3 times.DC is injection in the mouse tail vein.
Embodiment 5, cell in vitro cytotoxic activity detect
Carry out the cell in vitro cytotoxic activity with on-radiation lactic dehydrogenase enzyme process and detect, wherein, target cell and effector cell's preparation method is as follows:
1, the preparation of target cell: will count behind BST739 and the BST739-MUC1 cell dissociation, adjusting cell density is 2 * 10
5Individual/mL, add above-mentioned cell suspension 50 μ L in the every hole of 96 orifice plates, establish 4 multiple holes for every group.
2, effector cell's preparation: use the method identical to prepare spleen lymphocyte with embodiment 4.Lymphocyte is adjusted into different cell density (reduce serum-concentration and can reduce the substratum background) with complete 1640 substratum that contain 3% people AB serum.By imitating the effector cell that the target ratio is 1: 1,10: 1,50: 1,100: the 1 above-mentioned serial dilution density of adding 50 μ L in the hole of each target cell, cumulative volume is 100 μ L.
Mouse is contrast with (method is with aforementioned) after the ripe DC immunity of load M1 peptide three times with the DC+PBS immune group, and (series 1: target cell is BST739-MUC1 to detect splenic lymphocyte CTL effect; Series 2: target cell is BST739), the result is as shown in Figure 4.Imitating the target ratio is 50: 1 and 100: 1 o'clock, and mouse spleen lymphocyte has certain cellulotoxic effect to the BST739-MUC1 cell, but the molten broken rate of target cell is no more than 30%; Imitate the target ratio and be lower than 50: 1 obvious molten broken effects of nothing.All there is not the CTL effect when being BST739 with DC+PBS immunity or target cell.CTL is to be that target cell, effect target ratio are 50: 1 o'clock the molten broken rate of target cell with BST739-MUC1.
Observe the anti-tumour effect of M1 inducing peptide in embodiment 6, the tumor prevention model
24 of T739 mouse are divided into 3 groups at random, and 6 every group, carry out immunity (method is with aforementioned) with DC+M1 peptide, DC+TAT-M1 peptide and DC+PBS respectively, last immunity one week of back is at identical lymphoglandula drainage zone subcutaneous vaccination BST739-MUC1 cell 2 * 10
5Individual/only; Observed tumor formation rate on the 8th day; Went eyeball to get blood and execution in 16 days, carefully peel off tumour, weigh.Tumour and the tight person of skin adhesion are together taken off together with adhesion skin.Sample is with 4% formalin fixed, and routine pathology is cut into slices.Serum-20 ℃ preservation is used to detect M1 peptide specific antibody.Select a mouse at random for every group, under aseptic condition, get spleen and make single cell suspension, measure tumour size in the prophylaxis model, and carry out ELISA, ELISPOT and cytotoxic activity detection.Wherein, when the ELISA method detects, serum by dilution in 1: 100, is measured 450nm place absorbance, used two resist and are the goat anti-mouse igg of horseradish peroxidase-labeled (available from China fir Bioisystech Co., Ltd in Beijing), and chromogenic substrate is TMB (SIGMA).(1 is the DC+PBS immune group to tumour size comparison result as shown in Figure 5 in the prophylaxis model, 2 is DC+TAT-M1 peptide immune group, 3 is DC+M1 peptide immune group), concrete numerical value (n is the mouse quantity of this inspection) as shown in table 2, all the other every immunology detection results are also as shown in table 2, detected result can be brought out M1 specific cell and humoral immunoresponse(HI) after showing the DC immune mouse that the M1 peptide hatches, and can have certain lethal effect to the tumour cell of expressing MUC1 in vivo and in vitro.
Tumour size and immunologic test index result in table 3 prophylaxis model
| Tumor formation rate (%) n=6 | Heavy (mg) n=6 of knurl | ELISA n＝6 | ELISPOT(1/10 5) n＝1 | CTT(％) n＝1 | |
| DC+PBS DC+TAT-M1 peptide DC+M1 peptide | 100 67 67 | 11.8 4.8 1.7 | 0.57 1.47 1.52 | 3.8 41.8 131.5 | 7.8 13.8 17.3 |
Annotate: detect through the ELISA method, normal mouse serum OD450nm place absorbance is 0.009, and ELISPOT method detected result is with per 10
5The positive spots numerical table that individual spleen lymphocyte forms shows; CTL is to be that target cell, effect target ratio are 50: 1 o'clock the molten broken rate of target cell with BST739-MUC1.
Sequence table
<160>2
<210>1
<211>12
<212>PRT
＜213〉M13 phage (Phage M13)
<400>1
Lys His Tyr Asp Pro Phe His His Arg Met Pro Gln
1 5 10
<210>2
<211>36
<212>DNA
＜213〉M13 phage (Phage M13)
<400>2
aagcattatg atccgtttca tcatcggatg ccgcag
Claims (9)
1, the mucinous analogue epi-peptide of a kind of MUC1 is SEQ ID № in the sequence table: the polypeptide of 1 amino acid residue sequence.
2, the DNA of the mucinous analogue epi-peptide of the coding described MUC1 of claim 1.
3, DNA according to claim 2 is characterized in that: described DNA is SEQ ID № in the sequence table: 2 dna sequence dna.
4, the expression vector that contains claim 2 or 3 described DNA.
5, the transgenic cell line that contains claim 2 or 3 described DNA.
6, the host bacterium that contains claim 2 or 3 described DNA.
7, a kind of method of expressing the mucinous analogue epi-peptide of the described MUC1 of claim 1 is that the recombinant expression vector that will contain the mucinous analogue epi-peptide DNA of described MUC1 imports host cell, expresses obtaining the mucinous analogue epi-peptide of MUC1.
8, a kind of anti-tumor vaccine, its activeconstituents are the mucinous analogue epi-peptide of the described MUC1 of claim 1.
9, a kind of antitumor drug, its activeconstituents are the mucinous analogue epi-peptide of the described MUC1 of claim 1.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997011715A1 (en) * | 1995-09-27 | 1997-04-03 | The Austin Research Institute | Mimicking peptides in cancer therapy |
| WO2000073430A2 (en) * | 1999-05-27 | 2000-12-07 | Max-Delbrück-Centrum für Molekulare Medizin | Vaccines against conformation-dependent antigens and against antigens that are not or are not only proteins or peptides |
| CN1480463A (en) * | 2002-09-03 | 2004-03-10 | 吉林圣元科技有限责任公司 | Chirality peptide antigen and compon of bacterin based on core sequence of mucin 1 |
| CN1525864A (en) * | 2001-04-02 | 2004-09-01 | �Ϻ���ͨ��ѧ | Mucin peptides with immune enhancing properties |
-
2005
- 2005-06-21 CN CNB2005100776174A patent/CN1301267C/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997011715A1 (en) * | 1995-09-27 | 1997-04-03 | The Austin Research Institute | Mimicking peptides in cancer therapy |
| WO2000073430A2 (en) * | 1999-05-27 | 2000-12-07 | Max-Delbrück-Centrum für Molekulare Medizin | Vaccines against conformation-dependent antigens and against antigens that are not or are not only proteins or peptides |
| CN1525864A (en) * | 2001-04-02 | 2004-09-01 | �Ϻ���ͨ��ѧ | Mucin peptides with immune enhancing properties |
| CN1480463A (en) * | 2002-09-03 | 2004-03-10 | 吉林圣元科技有限责任公司 | Chirality peptide antigen and compon of bacterin based on core sequence of mucin 1 |
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